PROJECT SUMMARY/ABSTRACT (R35 GM124824) Chloride is the most abundant free anion in animal cells. It's not surprising that chloride channels are involved in a wide range of functions as diverse as cell volume regulation, epithelial fluid secretion, regulation of electrical excitability, and acidification of intracellular organelles. Their physiological function is impressively illustrated by many diseases (channelopathies) caused by chloride channel mutations, such as cystic fibrosis (1 in 2,000 Caucasians), myotonia, kidney stones, and osteopetrosis. However, despite recent progress, chloride channels are considerably under-studied compared to their cation (sodium, potassium, and calcium) channel cousins. Many electrophysiologically well characterized chloride channels still lack molecular identity. Several factors block progress in this field. Unlike cation channels, there are no sequence homologies (for example, conserved pore-lining motif) among known chloride channel families. The lack of specific high-affinity channel ligands (e.g. toxins) hinders direct purification. Expression cloning, an otherwise powerful technique, is hampered by high endogenous expression of channel channels in popular expression systems. The absence of molecular identity presents the biggest roadblock to elucidate the precise biological function of these widely expressed pore-forming membrane proteins. The proposed research program will combine increasingly powerful genomics tools (including bioinformatics, proteomics and gene manipulation) with electrophysiology and imaging techniques to identify novel chloride channels and investigate their physiological function using mouse models. Our results will shed light on the molecular identity and function of new chloride channels and may provide therapeutic strategies to target them for diseases with abnormal chloride transport and homeostasis.